Journal of Oleo Science
Copyright ©2007 by Japan Oil Chemists’ Society
J. Oleo Sci. 56, (5) 231-235 (2007)
NOTE
Sterol Composition in Muscle and Viscera of the
Marine Bivalve Megangulus zyonoensis from
Coastal Waters of Hokkaido, Northern Japan
Hideki Kawashima1＊, Masao Ohnishi2, Yukiko Negishi3,
Masao Amano4 and Mikio Kinoshita2
1
Bioscience Laboratory, Miyako College, Iwate Prefectural University (1-5-1 Ka-nan, Miyako 027-0039, JAPAN)
Department of Agriculture and Life Sciences, Obihiro University of Agriculture and Veterinary Medicine (Inada, Obihiro 080-8555, JAPAN)
3
Institute of Nutrition Sciences, Kagawa Nutrition University (3-9-21 Chiyoda, Sakado, Saitama 350-0288, JAPAN)
4
Department of Animal Sciences, Teikyo University of Science and Technology (2525 Yatsusawa, Uenohara, Yamanashi 409-0193, JAPAN)
2
Abstract: Sterol composition in muscle and viscera tissues of the marine bivalve Megangulus zyonoensis
was investigated. Among 13 different sterols identified by gas chromatography-mass spectrometry of their
trimethylsilyl ether derivatives, cholesterol was the most abundant sterol, followed by 24methylenecholesterol, in both muscle and viscera tissues. In this study, the proportion of brassicasterol, in
addition to that of cholesterol, differed between the muscle and viscera.
Key words: bivalve, Megangulus zyonoensis, sterol composition
1 INTRODUCTION
In Japan the marine bivalve genus Megangulus is a valuable food resource because of its soft flesh texture, rich
sweetness, and good taste. From the viewpoint of comparative biochemical interest, we previously analyzed various
tissues lipids of the marine bivalves Megangulus zyonoensis and Megangulus venulosus to compare the occurrence
and distribution of their fatty acid composition. The proportions of 20:5n-3 and 22:6n-3 were highest in muscle
lipids from the two Megangulus species, and the proportion of 22:6n-3 was much higher in their polar lipids of all
tissues of both these bivalves 1). Interestingly, nonmethylene-interrupted dienes (18:2 to 22:2) and trienes (20:3 and
22:3), whose physiological significance is currently
unknown, and the unusual tetraenes, 20:4n-1, 20:4n-4,
21:4n-5, and 21:4n-6, were found in the mantle, muscle and
viscera of M. zyonoensis in small amounts 2). In addition,
this bivalve was extremely rich in 19:1, 20:2, and 20:3 isomers, but the reason is not clear 2). In contrast to the occurrence and distribution of fatty acids noted above, those of
sterols in the marine bivalve M. zyonoensis have not been
characterized. Here, we provide the first data on the sterol
composition in muscle and viscera of this bivalve from
coastal waters of Hokkaido, northern Japan. Our results,
from this and previous studies, may contribute to fundamental research to develop a bivalve fishery, because our
knowledge of the occurrence and distribution of both fatty
acids and sterols in Megangulus species has been very little.
2 MATERIALS AND METHODS
The marine bivalve M. zyonoensis (shell length, 90-95
mm) was obtained from a local fish market (Tomakomai
Fishermen’s Cooperative Association, Hokkaido, Japan) in
August 2002. The muscle and viscera tissues of this bivalve
were dissected, suspended in a chloroform-methanol mixture (2:1, v/v), and then homogenized for 30 s at 891 × g
using an IKA ULTRA-TURRAX® T25 Basic (IKA Japan KK,
Nara, Japan) to disrupt cells. Lipids were extracted by the
method of Folch et al. 3) and saponified with 5% potassium
＊
Correspondence to: Hideki Kawashima, Bioscience Laboratory, Miyako College, Iwate Prefectural University, 1-5-1 Ka-nan, Miyako 0270039, JAPAN
E-mail: [email protected]
Accepted January 24, 2007 (received for review November 8, 2006)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://jos.jstage.jst.go.jp/en/
231
H. Kawashima, M. Ohnishi, Y. Negishi et al.
hydroxide in methanol at 90℃ for 1h. The reaction mixture
was cooled and extracted twice with dichloromethane. The
sterol fraction was purified by preparative thin-layer chromatography (TLC) on silica gel 60G (Merck, Darmstadt,
Germany). TLC plates were developed with n-hexane/
diethyl ether/acetic acid (90:10:2, v/v/v). The bands corresponding to identified sterols were scraped off the plates
and eluted with dichloromethane. The sterols were converted to their corresponding trimethylsilyl (TMS) ether
derivatives by the addition of 0.25 mL of BSTFA [N, O-bis
(trimethylsilyl) trifluoroacetamide]/TMCS [trimethylchlorosilane] (99:1, v/v) (Supelco, Bellefonte, PA) and heating at 70℃ for 1 h. Gas chromatography (GC) analyses
were performed using a Shimadzu GC-1700 instrument
(Shimadzu Corp., Kyoto, Japan) with a flame ionization
detector and a SAC TM-5 capillary column (30 m × 0.25 mm
i.d., 0.25 mm film thickness; Supelco, Bellefonte, PA). The
flow rate of the carrier gas helium was 0.9 mL/min, and
the injection split ratio was 1:50. The column temperature
was isothermal at 285℃. The injector and detector temperatures were 290 and 300℃, respectively. Capillary gas
chromatography-mass spectrometry (GC-MS) analysis of
free sterols and their TMS ether derivatives was performed using the same capillary column that was used for
GC analyses in a Hewlett-Packard 6890 series gas chromatograph connected to a 5973 N mass selective detector.
The carrier gas was helium, at a flow rate of 1.5 mL/min,
and pressure programming was used in a constant flow
mode. The oven temperature was isothermal at 300℃, and
the injection split ratio was 1:25. The temperature of the
interface between the GC and MS was held at 280℃. The
MS operating conditions were as follows: ion source temperature, 230℃; electron voltage, 70 eV; scanning range,
m/z 50-550. Identifications of the sterols in this study
were confirmed by comparison of their relative retention
time (RTT) values and their mass spectrometric fragments
with those of a known plant sterol mixture (Tama Biochemical Co. Ltd., Tokyo) and published data 4-8). Statistical analyses were conducted using Micro Excel Version 2004 for
Windows (Social Survey Research Information Co., Ltd.
Japan). Data are mean ± SD (n ＝ 3) in both text and Table
2. Statistical differences in mean values were assessed by
the Student’s t-test. A value of P ＜ 0.05 was considered
significant.
3 RESULTS AND DISCUSSION
The muscle and viscera of M. zyonoensis contained levels of sterols at 10.3 ± 1.8 and 13.6 ± 0.6%, respectively, of
total lipids. Thirteen different sterols, including three different stanols, were identified in the muscle and viscera by
capillary GC-MS of their TMS ether derivatives (Table 1).
As shown in Table 1, the major characteristic ions for D5-
sterols, detected in this study, contained molecular ion
[M]＋, [M-90]＋, [M-105]＋, [M-129]＋ and m/z 129 peaks 8).
Ten sterols (D5-sterols) and three stanols (5a-stanols) were
(numbered in order of increasing retention time): (1) 24-norcholesta-5,22E-dien-3b-ol (24-norcholestadienol); (2) cholesta-5,22Z-dien-3b-ol (cis-22-dehydrocholesterol); (3) cholesta-5,22E-dien-3b-ol (trans-22-dehydrocholesterol); (4)
cholest-5-en-3b-ol (cholesterol); (5) 5a-cholestan-3b-ol
(cholestanol); (6) 24-methylcholesta-5,22E-dien-3b-ol (brassicasterol); (7) 24-methylcholesta-5,24(28)-dien-3b-ol; (24methylenecholesterol); (8) 24-methylcholest-5-en-3b-ol
(campesterol) (9) 24-methyl-5a-cholestan-3b-ol (campestanol); (10) 24-ethylcholesta-5,22E-dien-3b-ol (stigmasterol);
(11) 24-ethylcholest-5-en-3b-ol (sitosterol); (12) 24-ethyl-5acholestan-3b-ol (sitostanol); and (13) 24-ethylcholesta5,24(28)-dien-3b-ol (isofucosterol).
The sterol composition of muscle and viscera of M.
zyonoensis is shown in Table 2. The relative proportions of
individual sterols ranged from ＜ 0.1 to 33% of total sterols.
Among 13 sterols identified in this study, cholesterol (2933% of total sterols) and 24-methylenecholesterol (one
fourth of total sterols) detected in both tissues were characteristically the major sterol components (＞ 10% of total
sterols), which were similar to those observed in the
bivalve Macoma balthica from the Baltic Sea 9). The proportion of major sterols in the muscle and viscera of M.
zyonoensis accounted for 58 and 55%, respectively. Interestingly, the proportions of 24-methylenecholesterol in
both tissues of M. zyonoensis were almost twice as high as
in M. balthica, but the proportion of cholesterol in M.
zyonoensis was lower than in M. balthica. Other sterols
identified in this study each accounted for less than 10% of
the total sterols. The sterol composition of M. zyonoensis
was very similar to that of the scallop species, Placopecten
magellanicus 10,11) and Pecten maximus 6), and the pacific
oyster Crassostrea gigas 12). By contrast with the major
sterols of M. balthica, the major sterols in these scallops
and the oyster are reported to be cholesterol, brassicasterol, 24-methylenecholesterol, and trans-22-dehydrocholesterol, which each accounted for more than 10% of
total sterols 6,10-12). However, brassicasterol, which is generally present in diatoms 13), and trans-22-dehydrocholesterod
levels each accounted for less than 10% of total sterols in
both tissues in this study. These differences may be mainly
due to variations in the sterol compositions of the diets
these bivalves fed on. The most interesting result was a
difference between the muscle and viscera of M. zyonoensis that was statistically characterized by the proportions
of brassicasterol and cholesterol. The proportion of cholesterol was significantly higher in the muscle than in the viscera, but that of brassicasterol was significantly higher in
the viscera. Several studies on the anatomical distribution
of sterols in marine bivalves showed insignificant variations in the sterol composition of different organs from a
232
J. Oleo Sci. 56, (5) 231-235 (2007)
Sterols of Megangulus zyonoensis
single individual or the same specimen 6,9,11).
Among 13 different sterols identified in this study, both
cis-22-dehydrocholesterol and trans-22-dehydrocholesterol
are present in the scallop P. magellanicus 10,11) , in the
gonad, muscle, and digestive gland of the scallop P. maximus 6), in the clam Ruditapes decussatus 14), in the mussels
Mytilus edulis and Atrina fragilis, and in the oyster
Ostrea edulis 15), but these sterol isomers may not have
been reported for other marine bivalves. Of particular note
was the presence of the 5a-stanols, cholestanol, campestanol, and sitostanol, which were found in both tissues of
M. zyonoensis in small amounts. Compared with sterols
widely distributed in marine bivalves, stanols other than
cholestanol can not be readily detected because of their
very small or trace amounts. Minor stanols in marine
bivalves may have originated from dietary microalgae or
sediments, or both. The occurrence and distribution of
campestanol and sitostanol, which are representative phytosterol components, in marine bivalves may not have
reported previously.
The biosynthesis and incorporation of sterols in a
marine bivalve of the genus Megangulus are still little
understood. Because the ability to synthesize or convert
sterols is generally low or absent in bivalves 11,15-17), a study
of the occurrence and determination of sterols in marine
bivalves provides valuable information on their nutrient
Table 1 Characteristic Mass Spectrometric Fragments of Trimethylsilyl Ether Derivatives of Sterols in the Marine
Bivalve Megangulus zyonoensis.
Sterol
Unsaturation
position
RRT a
Characteristic fragments (m/z, relative intensity)
24-Norcholestadienol
D5,22
0.706
442 (M+, 27), 427 (7), 352 (37), 337 (18), 313 (35), 255 (31), 215 (13),
129 (57), 97 (100)
cis-22-Dehydrocholesterol
D5,22
0.892
456 (M+, 38), 441 (9), 366 (51), 351 (22), 327 (43), 282 (6), 255 (50),
215 (16), 129 (80), 111 (100)
trans-22-Dehydrocholesterol
D5,22
0.920
456 (M+, 45), 441 (11), 366 (61), 351 (31), 327 (66), 282 (8), 255 (56),
215 (21), 129 (92), 111 (100)
Cholesterol
D5
1.000
458 (M+, 38), 443 (12), 368 (79), 353 (40), 329 (100), 247 (17), 213 (10),
129 (68)
Cholestanol
D0
1.014
460 (M+, 76), 445 (92), 403 (21), 370 (44), 355 (57), 305 (36), 230 (16),
215 (100)
Brassicasterol
D5,22
1.085
470 (M+, 51), 455 (11), 380 (63), 365 (25), 341 (38), 255 (65), 213 (19),
129 (95), 69 (100)
24-Methylenecholesterol
D5,24 (28)
1.220
470 (M+, 16), 455 (13), 386 (46), 380 (28), 365 (19), 343 (29), 341 (35),
296 (30), 281 (19), 257 (21),129 (100)
Campesterol
D5
1.235
472 (M+, 39), 457 (13), 382 (84), 367 (39), 343 (100), 315 (6), 289 (7),
225 (18), 213 (12), 129 (93)
Campestanol
D0
1.257
474 (M+, 59), 459 (73), 417 (20), 384 (30), 305 (25), 276 (13), 230 (19),
215 (100)
Stigmasterol
D5,22
1.312
484 (M+, 51), 469 (15), 394 (59), 379 (25), 355 (42), 255 (51), 213 (20),
129 (81), 83 (100)
Sitosterol
D5
1.473
486 (M+, 37), 471 (12), 396 (80), 381 (34), 357 (90), 329 (5), 303 (6),
275 (13), 255 (19), 213 (13),129 (100)
Sitostanol
D0
1.500
488 (M+, 40), 473 (51), 398 (25), 383 (33), 358 (12), 305 (19), 257 (19),
230 (21), 215 (100)
Isofucosterol
D5,24 (28)
1.526
484 (M+, 6), 469 (6), 386 (100), 371 (18), 355 (8), 296 (70), 281 (44),
257 (29), 255 (17), 213 (15),129 (68)
a
Retention times relative to cholesterol TMS ether derivative (15.61 min) using SACTM-5 capillary column (30 m × 0.25 mm i.d.,
0.25 mm film thickness; Supelco, Bellefonte, PA).
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J. Oleo Sci. 56, (5) 231-235 (2007)
H. Kawashima, M. Ohnishi, Y. Negishi et al.
Table 2 Sterol Composition (%) in Muscle and Viscera of the Marine Bivalve
Megangulus zyonoensis.
Muscle (n ＝ 3)
Sterol
a
24-Norcholestadienol
4.3 ± 0.0
cis-22-Dehydrocholesterol
1.9 ± 0.1
trans-22-Dehydrocholesterol
5.9 ± 0.3
Viscera (n ＝ 3)
5.7 ± 0.7b
2.1 ± 0.2
5.8 ± 0.1
Cholesterol
a
33.1 ± 1.0
29.3 ± 0.2b
Cholestanol
3.3 ± 0.4a
3.8 ± 0.4b
Brassicasterol
7.3 ± 0.4a
9.1 ± 0.8b
24-Methylenecholesterol
25.1 ± 1.0
25.5 ± 0.2
a
7.8 ± 0.3b
Campesterol
8.7 ± 0.3
Campestanol
0.7 ± 0.0
0.6 ± 0.3
Stigmasterol
＜ 0.1
1.6 ± 0.5
Sitosterol
4.5 ± 0.1a
Sitostanol
＜ 0.1
＜ 0.1
5.1 ± 0.1
4.9 ± 0.3
Isofucosterol
3.9 ± 0.2b
Values are mean ± SD of three independent experiments.
Values with different superscript letters in a column are significantly different (P ＜ 0.05) by
the Student’s t-test.
requirements and the efficiency of seed production in
hatcheries 11,18). Sterol composition may prove to be a useful
molecular biomarker, because bivalves incorporate the
sterols present in seawater - basically from microalgae and
sediments 19). Future studies are needed to determine if the
sterol composition of bivalves of the genus Megangulus is
related to their reproductive cycle, dietary habits and seasonal variations.
3.
4.
5.
ACKNOWLEDGEMENTS
This study was done as a cooperative research program
(no. 103) with the International Coastal Research Center,
Ocean Research Institute, The University of Tokyo. We
thank Mr. K. Omata, Tama Biochemical Co. Ltd., Tokyo, for
providing several authentic sterols.
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